Cells of Saccharomyces cerevisiue were loaded with indo-1, by incubation in a medium of pH 4.5, which contained pentapotassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-l was achieved. By perrneabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-l was accumulated in the cytosol. It was also shown that intracellular indo-l did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca*+ was found to be 346 nM. Vacuolar Ca*' concentration, calculated from indo-l fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.
Cytosolic Ca2+ concentrations ([Ca2+]i) were determined in haploid and diploid cells of Saccharomyces cerevisiae, loaded with indo‐1 and exposed to media containing a range of Ca2+ concentrations. [Ca2+]i homeostasis was maintained at the 100–150 nM level in cells which were pre‐incubated with glucose and exposed to O.1 μM–1O mM Ca2+ in the medium. Slightly higher levels of [Ca2+]i were determined in cells exposed to 50 mM Ca2+. Pre‐incubation with metabolic inhibitors instead of glucose caused a reduction in cellular ATP levels and an impaired [Ca2+]i homeostasis; [Ca2+]i reached 800 nM in cells exposed to 10 mM CaCl2. Cells of the Δvma4 mutant strain, with no functional vacuolar H1‐ATPase, had elevated levels of [Ca2+]i, reaching 1.8 μM when pre‐incubated with glucose and exposed to 10 mM CaCl2. Higher levels of [Ca2+]i were measured in the mutant cells which were pre‐incubated with metabolic inhibitors. This result indicates the central role of the vacuoles in maintaining [Ca2+]i‐homeostasis and suggests the presence of an additional non‐vacuolar ATP‐requiring mechanism which contributes to keeping [Ca2+]i at low levels.
Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organism's DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry.
Influx of 45Ca2+ into Sacckomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 rnin, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ infiux. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pHli, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pHli. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was Merent. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx. The role of the increase in cAMP level on Ca2+ influx was examined by measuring the effect of glucose and of artificial intracellular acidification on Ca2+ influx in a cyrl strain which lacks adenylate cyclase activity. In this strain, addition of glucose or isobutyric acid still led to a transient increase in Ca2+ transport. Therefore, we concluded that at least part of the increase in Ca2+ influx in response to glucose is CAMP-independent.
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